JP2017508148A - A predictive method for determining tissue radiosensitivity. - Google Patents

Abstract

A method for predicting cell radiosensitivity of a patient to ionizing radiation from cells sampled from a non-irradiated or slightly irradiated site of a patient, wherein (i) the sampled cells are amplified, The amplified cells constitute the “cell sample”, and (ii) the cell sample is observed using the marker pH2AX, observation time t4 after irradiation at 0 minutes (t0, non-irradiated state) and absorbed dose D The average number of nuclear lesions obtained at an observation time t is determined (this average number is defined as NpH2AX (t)), and (iii) the total dose that should not be exceeded (TDNTBE) expressed in gray (Gy) ) Is determined using at least the parameter NpH2AX (t4), where t4 is a fixed time representing the time taken for the DNA cleavage rate to reach its residual value. It is a constant value.

Description

  The present invention relates to the field of medical radiobiology, and more specifically to the field of radiobiological clinical laboratory methods. The present invention relates to a novel method for predicting cellular, tissue and clinical radiosensitivity based on the determination and correlation of multiple parameters and cellular enzymatic criteria.

  Tissue reactions (eg, dermatitis or proctitis) appear in about 1-15% of cancer patients undergoing radiation therapy. This tissue response can affect the treatment, and the physician may decide to stop radiation therapy before the planned protocol ends. The tissue reaction is also an indicator that the patient is particularly sensitive to ionizing radiation. Furthermore, radiation therapy can increase not only the morbidity of post-treatment complications, but even the patient's mortality, even if interrupted when the first visible histological signs appear. This is due to the fact that treatment was discontinued early and the cancer being treated could not be completely eradicated, and radiation itself caused incidental damage to healthy tissue.

  It is also known that the problem of tissue sensitivity to ionizing radiation cannot be separated from the problem of DNA damage repair mechanisms. In fact, at the cellular level, ionizing radiation can break multiple chemical bonds by generating free radicals (especially by overoxidation) and by generating other reactive species due to DNA damage. DNA damage caused by endogenous or exogenous stress (eg, ionizing radiation and free radicals) can be caused by base damage, single-strand breaks, and double-strand breaks, particularly depending on the energy applied. , DSB) can lead to various types of DNA damage. Unrepaired DSB is associated with cell death, toxicity, and more specifically radiosensitivity. Inadequately repaired DSBs are involved in genomic instability, mutagenic phenomena, and cancer predisposition. A living body has a specific repair system for each type of DNA damage. Mammals provide two major DSB repair modes: repair by suture (strand ligation) and repair by recombination (insertion of homologous or heterologous strands).

  It is known that the sensitivity of tissues to ionizing radiation varies greatly from organ to organ, and there are great individual differences. In 1981, Fertil and Malaise conceptualized “inherent radiosensitivity”. Therefore, various studies on the therapeutic effects and side effects of radiation therapy have shown that, in contrast to individuals with particularly high radiation resistance, from clinically recognizable but non-critical side effects to fatal side effects. It has been found that there are individuals with radiosensitivity. Except in certain rare cases with extreme radiosensitivity, whose genetic origin is likely to have been recognized, radiosensitivity is generally derived from genetic predisposition and is therefore considered to be unique to the individual. ing. For this reason, it is desirable to have a predictive testing method so that any patient can determine the maximum accumulated dose that can be received without risk. This problem occurs mainly in high ionizing radiation therapy. However, similar to those used for radiation therapy, all other types of exposure to strong ionization doses are likely to be problematic.

  Two proteins of the family of kinases, commonly referred to as ATM and ATR, are known to be involved in DSB detection, repair, and signaling. These actions require at least the presence of a protein known as BRCA1 and the presence of an ordered cascade of phosphorylations of various ATM substrates (N. Foray et al., “ A subset of ATM-and ATR-dependent phosphorylation events requires the BRCA 1 protein ”, TheEMBO Journal, 2003, vol. 22 (11), p.2860-2871). Attempts have been made to use ATM enzymes as an explanatory model for cell radiosensitivity (Joubert et al., “DNA double-strand break repair defects in syndromes associated with acute radiation response; At least two different assays to predict intrinsic radiosensitivity?”, Lnt J. Radiat. Biol. 2008, vol. 84 (2), p. 107-125), which enabled the identification of three types of radiosensitivity. Namely, cells with radiation resistance (Group I radiation sensitivity), cells with moderate radiation sensitivity (Group II radiation sensitivity), and cells with extremely high radiation sensitivity (Group III radiation sensitivity). . However, no prediction model based on this has been proposed. In particular, a relationship between clinical data (tissue severity) and radiobiological data has not been established. Similarly, N. Foray's presentation “Les reparatoses: nouveaux concepts sur la prediction de la radiosensibilite”, delivered during “Rencontres Nucleaire & Sante” on January 25, 2008 (XP55131242) The role of the different markers pH2AX and MRE11 and their changes over time has been suggested to explain the number of double-strand breaks caused, in this presentation quantifying and identifying the level of radiosensitivity observed at the clinical level There is no mention of tissue severity.

  The conditions under which ATM can contribute to the detection and repair of DNA damage are described in many references. WO 2004/013634 (KUDOS Pharmaceuticals Ltd) describes the identification of signaling pathways for ATM-dependent DNA damage that interact with other response factors including the MRE11 / Rad51 / NBS1 complex. US 2007/0072210 (Ouchi and Aglipay) proposes a method for screening potential therapeutic agents that promote response to DNA damage. In this method, a protein known as BAAT1 (related to a cancer predisposition associated with the BRCA1 gene), an ATM protein, and a candidate compound are mixed, and compared to a control mixture that does not contain the candidate compound, phosphorylation of ATM. The latter is identified as a potential active ingredient that promotes DNA repair. EP 2466310 (Helmholtz Zentrum Muenchen) describes the repair of double-strand breaks in DNA in the presence of a phosphorylated form of H2AX histone (called gamma-H2AX or g-H2AX). ing. WO 00/47760 and US Pat. No. 7,279,290 (St. Jude's Children's Research Hospital) describe the role of the kinase function of ATM in DNA repair.

  Thus, although these references describe the repair pathway, there is no suggestion of correlations that confirm clinical relevance.

  EP 1616011 (International Center for Genetic Engineering and Biotechnology) discloses a method for diagnosing genetic defects in DNA repair. This method is based on three steps: culturing cells isolated from the sample to be tested, incubating the cells with a chimeric polypeptide, and characterizing the cellular response. This cellular response is the expression rate of intracellular proteins, specifically biochemical markers consisting of p53, ATM, Chk1, Chk2, BRCA1, BRCA2, Nbs1, MRE11, Rad50, Rad51 type and histone. However, radiation-induced expression cannot predict the functionality of the protein (some syndromes show normal expression even if the protein is mutated). These procedures are not functional tests.

  WO 01/90408, WO 2004/059004, and WO 2006/136686 (French Atomic Energy Commission) describe methods for observing DNA damage resulting from ionizing radiation exposure. The first document discloses DNA damaging activity, but does not allow quantification of DNA removal and resynthesis enzyme activity or DSB repair. The other two references describe quantitative evaluation of DNA repair performance in biological media using superhelical circular double stranded DNA (according to the third reference, porous polyacrylamide hydrogels). Fixation in the membrane). These methods are not directly related to in situ DSB in a physiological environment and there is no correlation to evaluate the clinical application of these methods.

  In Korean Published Patent No. 20030033519, it is proposed to estimate radiosensitivity from catalytic activity or superoxide dismutase activity. In Korean Published Patent No. 20030033518, glutathione peroxidase or glucose 6-phosphate dehydrogenase is It is used. Such methods do not detect markers directly associated with DNA damage or repair.

  US Patent Application Publication No. 2011/31514 (Dana Farber Cancer Institute) proposes the use of FANCD2 lesion detection as a marker. US Patent Application Publication No. 2007/0264648 (National Institute of Radiological Sciences) proposes using DNA oligomers to predict the appearance of side effects during radiation therapy. However, some radiosensitivity can also be observed when the level of FANCD2 lesions is normal.

  US Patent Application Publication Nos. 2008/234946 and 2012/041908 (University of South Florida et al.) Describe a method for predicting the radiosensitivity of cancer cells rather than healthy cells. Although this is based on genomic data, it is not based on functional tests.

  WO 2014/154854 (Centre Hospitalier Universitaire de Montpellier) describes a method for predicting the radiosensitivity of a subject using at least one radiosensitive biomarker. This method does not detect markers directly associated with DNA damage or repair and is based on proteomic data. Furthermore, this patent application does not describe the quantitative relationship between the severity of radiobiological data and tissue response.

  WO 2013/187973 (University of California) describes a system and method for determining cell radiosensitivity and / or subject radiosensitivity relative to a control group. More specifically, the method includes irradiation of a biological sample and detection and quantification of radiation-induced lesions in erythrocytes, lymphocytes, and primary cells. These are results obtained from a blood sample using one or more detection markers selected from a set of markers including anti-pH2AX, anti-MRE11, and anti-ATM. By quantifying the radiation-induced lesion at each observation time (within 2 hours) after irradiation, the repair kinetics can be determined, which is experimentally correlated with the radiosensitivity of the subject. However, analysis of lesions in lymphocytes is very difficult because of their small nuclei. Furthermore, the method described above does not allow the physician to make decisions about the patient's treatment.

  U.S. Pat. No. 8,269,163 (New York University School of Medicine) describes the importance of accidental exposure to ionizing radiation for humans with the aim of ensuring that patients receive appropriate emergency treatment by triage. A number of proteins have been described that can be used as markers in simple and rapid evaluation. This patent relates to biological dosimetry (determining incidental dose), while radiosensitivity is detected using known doses.

  WO 2010/88650 (University of Texas) describes methods and compositions for identifying cancer cells that are sensitive or resistant to specific radiation treatments. Therefore, this document is not applicable to all radiotherapy.

  WO 2010/136842 (Philips) describes a comprehensive method for monitoring patients during radiation therapy using biomarkers. The method includes obtaining at least one descriptor from an image extracted from an imaging device. This descriptor belongs to a tissue of interest for radiation therapy or a tissue in the vicinity of the target site. The method further includes selecting at least one disease specific biological marker capable of detecting or quantifying radiation treatment side effects in the tissue region of interest. The method further includes collecting at least one in vitro measurement of a biomarker specific to the selected disease. Further, the method includes processing at least one descriptor of the in vitro measurement value of the at least one biomarker by a correlation technique, and outputting a signal indicating radiotoxicity in the tissue region of interest. However, the teachings of this patent only consider tissue-dependent toxicity, not individual toxicity, and are primarily based on image analysis.

  WO 2010/109357 plans an applicable radiotherapy protocol based on optimizing the probability of complications in normal tissue and the probability of tumor control according to markers specific to each patient Methods and apparatus for doing so are described. Marker values associated with normal tissue include in vitro test values, protein mass spectrometry signatures, and patient historical data. In vitro test values may be of cellular, proteomic, and genetic origin and include various cell counts, HB, CRP, PSA, TNF-alpha, ferritin, transferrin, LDH, IL-6, hepcidin , Creatinine, glucose, HbAlc, and telomere length, and the like. Patient history markers include measurements related to previous abdominal surgery, administration of hormonal drugs or anticoagulants, diabetes, age, and tumor growth. Biomarkers that are not associated with radiation toxicity are also envisioned, such as biomarkers associated with various ablation or chemotherapeutic agents. However, individual radiosensitivity is not considered.

  In spite of the existence of such a variety of prior art, the applicant has allowed the above-mentioned patent to assess the risk of tissue reaction after radiation treatment, and for any patient to use DSB. It was noted that no predictable method of quantifying an individual's radiosensitivity that could be used with any type of ionizing radiation that could be triggered was described. That is, there is still no effective solution to the problem of providing a method for predicting an individual's radiation sensitivity. The present invention aims to provide a novel method for predicting tissue and clinical radiosensitivity.

  The inventors obtained the following knowledge, and the method of the present invention was devised from this knowledge. That is, DNA double-strand breaks (DSB) represent the type of radiation-induced damage that best predicts radiosensitivity when it is not repaired, and best predict genomic instability when repair is inadequate It is a finding that represents the type of radiation-induced damage. The inventors have discovered that DSB is addressed by a major repair mode called joining and / or by a minor and incomplete repair mode called MRE11-dependent recombination. These repair modes are controlled by ATM proteins. The marker pH2AX indicates DSB recognized by the binding repair mode. Marker MRE11 indicates a DSB site that has been treated with incomplete MRE11-dependent recombination. The marker pATM provides information on the activation of the binding pathway by phosphorylation of H2AX and inhibition of the MRE11 dependent pathway.

  The inventors have also observed that the cytoplasmic form of ATM protein migrates into the cell nucleus after oxidative stress, particularly stress related to ionizing radiation that induces DSB.

  In order to observe DNA damage due to exogenous stress, it is necessary to consider both the natural state of DNA and the state caused by radiation. Furthermore, DNA repair needs to be taken into account after irradiation, but the kinetics of this DNA repair depends on the repair mechanism and therefore on the type of radiation-induced damage. Furthermore, it is known that there are individual differences in the effectiveness and speed of DNA repair, and that certain genetic conditions exist that lead to exceptional radiosensitivity.

  According to the present invention, the above problems are related to 1) amplification of cells of non-transformed cells, particularly skin biopsy materials, 2) effective mechanistic models for human resting cells, and 3) how to treat Regardless, it is solved by a method based on DSB recognition, repair, and signaling functional testing that is valid.

The first subject of the present invention is a method for predicting cell radiosensitivity to ionizing radiation of a cell sample obtained from cells sampled from a non-irradiated or slightly irradiated part of a patient . In this method,
(i) the sampled cells are amplified, and the amplified cells constitute the “cell sample”;
(ii) For the cell sample, using the marker pH2AX, the average number of obtained nuclear foci is determined at the observation time t (this average number is _defined as N _pH2AX (t)), and the observation time t is 0 Minutes (t0, non-irradiated state) and t4 (and preferably t1, t2, and t3) after irradiation with absorbed dose D,
(Iii) a total dose not to be exceeded (TDNTBE), expressed in gray (Gy), is determined using at least the parameter NpH2AX (t4);
(Iv) For the cell sample, using at least two of the markers pH2AX, pATM, and MRE11, the average number of nuclear foci is determined at the observation time t, and these average numbers are respectively determined as N _pH2AX (t ), N _pATM (t), N _MRE11 (t), and the observation time t is 0 minutes, that is, t0 representing a non-irradiation state, and observation times t1, t2, t3, and t4 after irradiation with the absorbed dose D At least one selected,
(V) a radiosensitivity group of the sample is determined using at least the average number N _pH2AX (t), N _pATM (t), and N _MRE11 (t);
The t4 is a fixed value indicating the time required for the level of DNA cleavage to reach its residual value, and preferably 6 to 8 times t3, but 12 hours or more, preferably 12 Time to 48 hours, more preferably about 24 hours,
The above-mentioned t3 is a fixed value representing the time at which about 25% of the DSB of the control cells collected from a patient having radiation resistance is repaired at the end thereof, and preferably 3 to 5 times t2. However, it is selected to be 2.5 hours or more and 6 hours or less, preferably 3 hours to 5 hours, more preferably about 4 hours,
The t2 is a fixed value representing a time at which about 50% of the DSB of the control cells collected from a patient having radiation resistance is repaired at the end, and preferably 5 to 7 times t1. However, it is selected to be 35 minutes or more and 90 minutes or less, preferably 45 minutes to 75 minutes, more preferably about 60 minutes,
t1 is a fixed value representing the time at which the number of recognized DSBs reaches the maximum value in the control cells collected from the patient having radiation resistance at the end thereof, preferably 5 times from the stop of irradiation. Minutes to 15 minutes, preferably 7.5 minutes to 12.5 minutes, more preferably about 10 minutes.

  In a preferred embodiment, the average number of nuclear lesions obtained at observation times t1, t2, and t3 using the marker pH2AX is used to verify the DSB dynamic recognition shape and repair curve.

  The total dose that should not be exceeded (TDNTBE), expressed in gray (Gy), is an important parameter for the radiation therapist, which allows any patient to absorb without causing a potentially fatal response Can predict dose. This parameter can also eliminate radiation therapy for patients with particularly high radiosensitivity.

  TDNTBE is ideally represented as a skin equivalent. Since the test is performed on dermal fibroblasts, TDNTBE cannot be quantitatively extrapolated directly to other tissues. As such, the prediction is substantially related to the radiation-induced response of skin or biologically equivalent tissue (eg, lung fibroblasts). However, extrapolation to other tissues such as endothelium, astrocytes and epithelium may be performed qualitatively. Nonetheless, a more accurate definition of corrective factors specific to each organization is awaited.

According to the present invention, TDNTBE is
If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / N _pH2AX (t4)], or
If N _pH2AX (t0)> 3, it can be determined by the formula: TDNTBE = 60 / [N _pH2AX (t4) + N _pH2AX (t0)].

In a variation of the method of the present invention, for the cell sample, the average number of nuclear lesions (expressed in%) observed for 100 cells at time t is also determined (this average number is N _MN (t)). ), Time t is at least t4 after irradiation with t0 (non-irradiation) and absorbed dose D, and the parameter N _MN (t4) is used to determine the TDNTBE. However, the statistical uncertainty of experimental micronucleus measurements is higher than the statistical uncertainty of the number of nuclear foci observed with immunofluorescence, so the predictive value of lesion measurements is the prediction of micronuclei. It may be said that it is preferable to the numerical value.

Therefore, for example, TDNTBE can be determined based on N _MN (t) by the following equation.

If N _pH2AX (t0) ≦ 3, the formula: TDNTBE = 60 / [0.4 × N _MN (t4)], or if N _pH2AX (t0)> 3, the formula: TDNTBE = 60 / [2+ ( 0.4 × N _MN (t4))]
Using one or the other of the method variants of the invention results in a decimal. The final TDNTBE value obtained after use of one or the other of the above formula is an integer corresponding to the arithmetically rounded value obtained by the calculation. TDNTBE determined by the present invention corresponds to this integer.

  According to one variant of the method of the invention, preferably for patients belonging to the type II radiosensitivity group (medium radiosensitivity), for the purpose of assigning an accurate TDNTBE to the patient, A radiosensitivity group is determined before the TDNTBE.

The radiosensitivity of the sample is determined by the following method according to the above formula.
(A) N _pH2AX (t4) <2, _NpATM (t1)> _NpATM (t2), _NpATM (t1)> 30, A <10, B <5, and C <2, provided that
C = N _pH2AX (t0) + N _MN (t0);
B = percentage of macronuclei (nuclei greater than 150 μm ² ) at t0;
When A = N _MRE11 (t0) + pH 2AX small lesions per cell at t0, the sample is considered to be radioresistant,
(B) when (N _pH2AX (t4)> 8, or N _MN (t4)> 24), the sample is considered to have high radiosensitivity;
(C) For all other conditions, the sample is considered to have moderate radiosensitivity.

For some patients, DNA double-strand breaks (DSBs) occur spontaneously and continuously due to the high frequency recombination phenomenon commonly found in patients susceptible to cancer. Repair can be inhibited. DSB spontaneous overproduction can have two consistent effects. That is, in the natural state, labeling with pH2AX can result in the appearance of nuclear foci smaller than the normally seen pH2AX foci, but these reflect the presence of numerous DSBs (the “small foci” phenomenon). Similarly, overproduction of SSB can lead to chromatin decondensation, which leads to an increase in cell nuclei (generally larger than 150 μm ² ; corresponding to a “macronuclear” phenomenon). These two phenomena reflect the high genomic instability.

  Next, TDNTBE is determined for the patients of radiation sensitivity group II through the above two modifications. Specifically, it is as follows.

If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / N _pH2AX (t4)], or
If N _pH2AX (t0)> 3, it is determined by the formula: TDNTBE = 60 / [N _pH2AX (t4) + N _pH2AX (t0)].

Alternatively, according to a second modified example, the formula: a, based on the _N MN (t) TDNTBE, can be determined by the following equation.

If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / [0.4 × N _MN (t4)] or
If N _pH2AX (t0)> 3, it can be determined by the formula: TDNTBE = 60 / [2+ (0.4 × N _MN (t4))].

  Using one or the other of the method variants of the invention results in a decimal. The final TDNTBE value obtained after using one or the other of the above formulas is an integer corresponding to an arithmetically rounded value obtained by calculation. TDNTBE determined by the present invention corresponds to this integer.

  The method of the present invention uses at least one healthy tissue sample, preferably fibroblasts. The fibroblasts are preferably sampled from the patient's connective tissue. This sampling may be performed by biopsy. Thus, in a preferred embodiment, the sampled cells are fibroblasts taken from a patient's skin biopsy (typically according to a method known as “skin punch biopsy”). ) The tissue sample is cultured in a suitable medium.

  In the first step of the method of the invention performed after cell sampling (specifically, in the preferred embodiment, through establishment of a biopsy of a fibroblast cell line), the natural state of the DNA (the state at t0) In other words, the characteristic of the state which does not receive irradiation is specified. This process may include, inter alia, investigating the size of the nucleus, the presence of micronuclei, spontaneous apoptotic bodies and multiple cells with multiple damages. Cells are observed with a fluorescence microscope. Using the DAPI dye (4 ', 6'-diamidino-2-phenylindole, CAS No. 28718-90-3 for dihydrochloride), the micronucleus per 100 cells is an indicator of genomic instability The level is determined. Apoptotic bodies are also determined. An abnormally large population of nuclei whose presence is indicative of chromatin decondensation is also determined.

  Ionizing radiation is defined by the absorbed dose (referred to as D, a parameter expressed in gray). According to the present invention, the absorbed dose D is 0.5 Gy to 4 Gy, preferably 1 Gy to 3 Gy, more preferably 1.7 Gy to 2.3 Gy, and even more preferably 2 Gy. These ranges typically correspond to individual radiotherapy sessions, the number of sessions depending on the site, and the type and condition of tumor progression.

  In the method of the present invention, all time values t1, t2, t3, and t4 are taken at the start of a series of tests (ie, for at least one arbitrary patient, preferably the method is statistically significant). In order to calibrate for a group of observations, it is important to define (for multiple patients) and that these time values be the same for all parameter determinations representing the time interval.

  In the method of the present invention, t1 is preferably from 8 minutes to 12 minutes, and / or t2 is preferably from 50 minutes to 70 minutes, and / or t3 is preferably 3. 5 hours to 4.5 hours and / or t4 is preferably 22 hours to 26 hours. It is preferred that all four conditions are satisfied.

  In a particularly advantageous and simple standardization variant of the method according to the invention, t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours and D is 2 Gy.

Preferably, the determination of N _pH2AX (t) involves an immunofluorescence test.

  Control cells from patients with radioresistance can be selected through clinical examination as cells from patients selected as patients who did not show a significant tissue response during or after radiation therapy. The control cells may also be selected as cells whose in vitro clonogenic viability shown after irradiation with an absorbed dose of 2 Gy is higher than 55%.

  Hereinafter, typical embodiments will be described.

Cells are observed using the marker pH2AX. Observations at the observation time t using the markers pATM and / or MRE11 (the average numbers of these are referred to as N _pATM (t) and N _MRE11 (t), respectively) and t1 after irradiation with the absorbed dose D, An observation at at least one observation time selected from t2, t3 and t4 can also be added. In one embodiment, the number of lesions is determined by the marker pH2AX and the presence of multiple lysed cells. The position of the pATM protein and the position of the MRE11 protein (nuclear or cytoplasmic) are recorded.

  This first step can identify potential genomic instabilities in the natural state.

  The second step of the method of the present invention involves irradiation with a desired absorbed dose D (eg 2 Gy) and evaluation of the cellular response to ionizing radiation.

  a) In the first embodiment, the repair by the joining of radiation-induced DSB is examined. Binding is the primary repair mode of DSB. The number of pH2AX lesions per cell is determined at t4, and further determined at t1 and t2 as necessary, and also at t3. By the determination at t3, the definition of the dynamic speed from t1 to t4 can be strengthened. In a preferred embodiment, after t4, the level of micronuclei is also determined to estimate the level of radiation-induced micronuclei. Thereby, the radiosensitivity can be estimated based on the superiority of the unrepaired DSB.

  b) In a second embodiment, a more detailed investigation of the cellular response to ionizing radiation is performed through measurement of the functionality of ATM-dependent kinase activation. In radioresistant control cells, the phosphorylated form of ATM protein (pATM) is known to be cytoplasmic in nature. Applicants have discovered that in the post-irradiation state, the phosphorylated form of the ATM protein tends to be nuclear. Once transferred to the nucleus, the pATM type activates the repair mechanism by binding and inhibits the incomplete MRE11-dependent repair pathway.

  As an example, if the pATM type shows cytoplasmic localization after irradiation (eg, absorbed dose 2 Gy), it can be concluded that the pATM type does not migrate from the cytoplasm to the nucleus or cannot migrate normally. This may be due to a mutation in the ATM, or a mutation in either the partner protein of the ATM that assists the ATM in moving to the nucleus after irradiation. In any case, this is a very high radiation sensitivity.

  The position determination of the pATM protein is performed at least at t1 and t2, and is also performed at t3 and t4 as necessary.

  c) The third embodiment can be combined with the above embodiment, expanding the investigation of cellular responses to ionizing radiation via MRE11-dependent pathways. For the main repair pathway by binding, its ability can be quantified by pH2AX immunofluorescence, but in addition to this repair pathway, Applicants have identified another repair pathway. This alternative repair pathway is an alternative to binding and is used instead of binding in the case of a gene defect. This is a repair by MRE11-dependent recombination. The ability of this pathway can be quantified by kinetic studies of immunofluorescence testing of MRE11 lesions. This measurement is performed at least at t1, t2, and t3, and is also performed at t4 as necessary. According to applicants' knowledge, in a radiation resistant control strain, MRE11 is in the cytoplasm and the number of MRE11 lesions is very small (typically MRE11) up to 4 hours after a dose of 2 Gy. The number of lesions is 7 ± 2). About 24 hours after irradiation, the marking is intracellular.

  In the last step, the results are evaluated by score calculation in order to predict the state of radiation-induced damage and / or the patient's radiosensitivity, in particular the patient-specific TDNTBE.

  FIGS. 1A, 1B, and 1C respectively show (A) change in the number of micronucleus lesions from non-irradiated cells, (B) change in the number of markers pH2AX, and (C) change in the number of markers pATM in the CTCAE classification. Shown in relation to severity along. Radiosensitivity cannot be predicted from non-irradiated cell micronuclei, pH2AX lesions, and pATM lesions.

  There are two different measures for the severity of tissue reaction, namely the CTCAE classification and the RTOC classification.

  The so-called CTCAE (Common Terminology Criteria for Adverse Events) classification is an indication of adverse events (especially side effects) in cancer treatment, published in 2006 by the National Cancer Institute of the United States of America. It is a term standard.

  An adverse event is any adverse, unintentional sign, symptom, or illness that is temporally related to the use of medical treatment or medical treatment and is considered to be related to that treatment or medical treatment. May or may not be considered. An adverse event is a unique representation of a specific event that is used for medical document management and during scientific analysis.

  In CTCAE, each adverse event is briefly defined for the purpose of clarifying the meaning of the adverse event. This scale is also effective for other genotoxic stresses (eg, burns), but is particularly used in radiation therapy.

  The grade indicates the severity of the adverse event. There are five severity grades (1-5) of CTCAE, with a unique clinical explanation for the severity of each adverse event. This is shown in Table 1 below. Each severity grade is defined by a specified tissue response.

  To these five grades, grade 0 corresponding to no influence on the structure is added.

  The so-called RTOG classification, a historical classification proposed by the Radiation Therapy Oncology Group (RTOC) in 1984, substantially covers all types of toxicity that occur after radiation therapy. Yes.

  However, the RTOG classification is not applicable to certain types of cancer, while the CTCAE classification is used for all types of cancer.

  2A and 2B show the change in the number of micronuclei 24 hours after irradiation in relation to the severity grade of CTCAE (FIG. 2A) or RTOG (FIG. 2B). Micronuclei were labeled using DAPI fluorescent markers and then quantified by fluorescent signal analysis. In FIG. 2, the radiosensitivity groups (I, II, III) are indicated by Roman numerals.

  Only group III radiosensitivity can be predicted by the number of micronuclei 24 hours after irradiation.

  3A, B, and C show the change in the number of pH2AX lesions 24 hours after irradiation in relation to the severity grade of CTCAE (FIG. 3B) or RTOG (FIG. 3C). FIG. 3A shows the time course of the average number of lesions obtained using the marker pH2AX.

  The radiosensitivity of group I, II, or III can be predicted by the number of pH2AX lesions obtained in association with the severity grade of CTCAE or RTOG (two different severity scales of tissue response) 24 hours after irradiation. However, the severity grade cannot be predicted.

  FIG. 4A shows the average number of lesions obtained using the marker pH2AX 10 minutes after irradiation with 2 Gy for all cell lines of collected patient samples (skin fibroblasts). The dotted line shows the normal incidence of DSB, ie 40 DSBs per Gy per cell.

  FIG. 4A shows that all cells from patients with Group II radiosensitivity are characterized with less pH2AX foci (DNA double-strand breaks (DSB)) than expected after 2 Gy. This is explained by the fact that DSB is only poorly recognized.

  FIG. 4B shows pATM expression in the cytoplasm and in the nucleus at each time (0 minutes, 10 minutes, and 1 hour) after irradiation with a dose of 2 Gy. Corresponding immunofluorescence data are shown for non-irradiated cells and cells 10 minutes after 2 Gy irradiation.

  The data shown in FIG. 4B is related to the number of pATM lesions and suggests an ATM “cyto-nuclear transit”.

  FIG. 4 (C) shows the dynamics of the average number of lesions obtained over time based on the collected cells using the marker pATM. For convenience, error bars associated with measurements performed at 10 minutes, 1 hour, 4 hours, and 24 hours were omitted. In FIGS. 4A and 4C, each point represents the average of three separate iterations, and the error bars represent the standard deviation for each category.

  FIG. 5 shows the change in the number of pATM lesions 10 minutes after 2 Gy irradiation (FIG. 5A) and the change in the number of pATM lesions in 1 hour (FIG. 5B) in relation to the CTCAE severity grade. Show. FIG. 5C shows the maximum number of pATM lesions between the two values obtained at 10 minutes and 24 hours after 2 Gy irradiation shown in FIGS. 5A and 5B, respectively, in relation to the CTCAE severity grade. Show.

  FIG. 5B shows grade 0, ie no impact on the tissue.

  FIG. 5C shows the relationship between radiobiological parameters and severity grade according to CTCAE classification for 100 patients. That is, FIG. 5C shows a clinical assessment of the correlation that exists between the CTCAE severity grade and the maximum number of pATM lesions between the two values obtained 10 minutes and 1 hour after 2 Gy irradiation. Represents.

  In FIG. 5, the radiosensitivity groups (I, II, IIIa and IIIb) are indicated by Roman numerals. For FIGS. 5A, 5B, and 5C, each point represents the average of three separate iterations in each category.

  The maximum number of pATM lesions in (2Gy + 10 minutes) or the maximum number of pATM lesions in (2Gy + 1 hour) can predict not only the severity grade of the response, but also all groups.

  FIG. 6A shows the maximum number of lesions obtained using the marker pATM in relation to the number of pH2AX lesions previously shown in FIGS. 3B and 5C.

  FIG. 6B shows the same data as that of FIG. 6A, with clearly defined confidence intervals representing the various human radiosensitivity groups (Group I, Group II, and Group III). Radiosensitivity is determined by recognition and repair of double-strand breaks.

  FIG. 6C shows the number of group occurrences for each group type. The probability of belonging to an arbitrary group is considered to be proportional to the inversion of the corresponding confidence interval, and the normalization frequency of each group is represented by a bar graph in FIG. 6C. The dotted line corresponds to the Gaussian producing the best data adjustment (r = 0.9).

Description of the invention
A. General definitions The terms “radiation-induced damage”, “radiation-induced”, “radiation sensitivity”, “radiation resistance”, “radiotoxicity”, and “radiotherapy” are all ionizing radiation, especially alpha (α) particles Or particle radiation consisting of beta (β) particles, or high energy electromagnetic radiation, in particular gamma (γ) radiation or X-ray radiation.

The term “ATM cell-nuclear translocation” refers to the movement of ATM proteins from the cytoplasm to the nucleus, particularly after irradiation.
B. DETAILED DESCRIPTION Hereinafter, embodiments having a plurality of modifications will be described. This embodiment is suitable for human patients.
1. Prior to sample preparation and manipulation of the sampled cells, each operator (eg, belonging to a cytological analysis laboratory) may be transferred to the patient's HIV or hepatitis C (typically from a physician). Get notified about the possibility of infection. The purpose of this notification is to allow the operator to take appropriate biological safety measures during sampling, operation, and cell culture management.

  The operator then takes a cell sample from the patient. The operator preferably takes a sample by biopsy of a skin sample. This sampling can preferably be performed by a method known as “skin punch biopsy”. Cell samples are placed in DMEM sterile medium (sterile fetal bovine serum DMEM + 20% medium) supplemented with 20% fetal bovine serum. Immediately move the sample to a specialized laboratory. This is because the sample should not be left at room temperature for more than 38 hours.

  Once transferred to the laboratory, a cell sample (typically a biopsy) is established as an amplifiable cell line without the use of viral or chemical conversion agents. This procedure is performed according to auxiliary procedures known in the culture laboratory, as highlighted in the publication “The radiobiology of cultured mammalian cells” by Elkin M. et al. (Gordon and Breach (1967)). When the number of cells is sufficient (1-3 weeks), the first test is performed using the method of the present invention. Cells are seeded onto glass slides in petri dishes. A part of the slide glass is irradiated with an absorbed dose D (for example, 2 Gy) in a medical irradiator according to an approved dose measurement method. The other slide glass is not irradiated and represents a natural state (absorbed dose is 0 Gy).

Irradiation can be performed using, for example, a medical accelerator that emits 6 MV photons at an absorbed dose rate of 3 Gy min ⁻¹ . After irradiation, the cells are maintained at 37 ° C. in a culture incubator in order to pass the repair time described below.

  For irradiated cells, features corresponding to the radiation-induced state are obtained after multiple repair times (post-irradiation repair times). Preferably, at least two time points are obtained, more preferably at least three time points, in particular t1, t2, t3 and t4. The above features are represented by lesions corresponding to the marker pH2AX.

The cells on the glass slide are then fixed, lysed and hybridized. This can be done using known procedures (see cited Bodgi et al. Publication). Specifically, cells were fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature and 20 mM pH 7.4 HEPES buffer (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid ), 50 mM of NaCl, 3 mM of MgCl _2, 300 mM sucrose, 0.5% Triton X-100 (formula _{t-Oct-C 6 H 4} - (OCH 2 CH 2) xOH ( wherein x = 9 to 10) And a nonionic surfactant represented by the formula, CAS No. 9002-93-1, manufactured by Sigma Aldrich) for 3 minutes. The cover slide was then washed in phosphate buffered saline (known as PBS) prior to immunological staining. Incubation was carried out at 37 ° C. for 40 minutes in PBS supplemented with 2% bovine serum albumin (known by the acronym BSA or fraction V; Sigma Aldrich). After incubation, washing was performed with PBS. Anti-pH2AX primary antibody was used at a concentration of 1: 800, and other primary antibodies were used at a concentration of 1: 100. Incubation with anti-mouse secondary antibody FITC or anti-rabbit secondary antibody TRITC (1: 100, Sigma Aldrich) was performed in 2% BSA at 37 ° C. for 20 minutes. To label the nuclei, the glass slides were treated with Vectashield ^™ containing DAPI (4,6-diamidino-2-phenylindole). Staining using DAPI is also indirectly in the number of cells in phase G ₁ (cells with uniform DAPI staining), the number of cells in phase S (cells with many pH2AX lesions), and phase G ₂ It enables determination of the number of cells (cells with non-uniform DAPI staining) and metaphase (state where chromosomes are visible).

Results are obtained from these slides using an immunofluorescent microscope (eg Olympus model). Reading may be direct (typically counting lesions of at least 50 cells at each point in G ₀ / G ₁ ) or using dedicated image analysis software Alternatively, an automatic microscope may be used. Software or automated microscopy methods are preferably calibrated manually.

To obtain results with sufficient statistical confidence to use as a basis for diagnosis, perform at least three separate series of tests (irradiation) and calculate the average number of each lesion for a specified time .
2. Biological and clinical parameter determination
2.1 Overview and Markers Used The present invention is based on the use of data obtained for unirradiated cells (natural state) and irradiated cells (radiation-induced state), especially for the marker pH2AX. The method of the present invention is based on investigating the kinetics of labeling with the markers in relation to the duration of repair. That is, the sample is labeled after a specified time since the cessation of irradiation and the sample's immunofluorescence is examined. For example, a total dynamic curve represented by five points located at t0, t1 (preferably 10 minutes), t2 (preferably 1 hour), t3 (preferably 4 hours), and t4 (preferably 24 hours). It can be measured. Here, t0 is a state before irradiation (natural state). Preferably, the data obtained using the other two markers, specifically pATM and MRE11, are related.

  However, Applicants have found that certain points (corresponding to certain repair times) are more important than others, and certain points are not predictive. By appropriately selecting the parameters determined at any time, the number of measurements can be reduced, thus reducing the overall cost of diagnosis without reducing the predictive performance of the method. This simplified method forms the basis of the predictive method of the present invention.

  If sampling is n = 3 (not Gaussian standard error SE), the average of each point and each dose with each marker is calculated using the standard error of the mean (SEM).

  (I) pH2AX refers to the phosphorylated form of serine 439 in the X variant of histone H2AX and, according to Applicants' knowledge, labels the number of DNA double-strand breaks (DSB). DSB is recognized by a major repair mode, namely binding. In effect, the marker pH2AX is in the nucleus exclusively in the form of nuclear lesions, and only the number and size of the lesions are analyzed.

  (Ii) pATM refers to the phosphorylated form of serine 1981 of the ATM kinase protein. According to the applicant's knowledge, ATM moves from the cytoplasm to the nucleus after irradiation under normal conditions (radiation resistant state). pATM is concentrated primarily in the cytoplasm and labels the DSB site. The marker pATM is characterized by localization, which can be either uniform cytoplasmic localization without nuclear foci (no cytoplasmic foci) or localization in the nucleus exclusively in the form of nuclear foci (homogeneous nuclei). May be cytoplasmic and nuclear lesions.

  (Iii) MRE11 is an endonuclease that cleaves DNA. According to Applicant's knowledge, MRE 11 marks DSBs that are poorly repaired upon completion of the repair process. The marker MRE11 is in the cytoplasm without a lesion, in the cytoplasm and nucleus without a lesion, or in the cytoplasm and nucleus with a lesion.

Counterstaining with DAPI (a DNA marker known to those skilled in the art) allows localization of the nucleus for localization of cytoplasmic or nuclear localization (this distribution of ionizing radiation Under effect, changed for MRE11 and pATM). The purpose of this is to quantify the size of micronuclei, apoptotic bodies, and nuclei, which are complementary cell markers to data about lesions.
2.2 Biological parameters The following items are defined and determined as follows:
N _pH2AX (t), _NpATM (t), N _MRE11 (t): Observation after observation time t0 (non-irradiation) or radiation irradiation (absorbed dose: 2 Gy) using markers pH2AX, pATM, and MRE11 Average number of nuclear lesions obtained at times t1, t2, t3, t4. However, while the determination of the parameter N _pH2AX (t) is indispensable in the method of the present invention, the determination of the other parameters N _pATM (t) and N _MRE11 (t) is arbitrary but suitable.
The number (%) of micronuclei observed per 100 cells in a natural state (t = t0, ie, no irradiation) or t = t4 after irradiation with an absorbed dose of 2 Gy.
2.3 Predictive evaluation The objective is to predict clinical or radiotherapy parameters based on measured biological data. This includes quantitative analysis, which is based directly on mathematical values of scores or mathematical formulas that relate the scores. This analysis relates to the total dose that should not be exceeded (a criterion called TDNTBE) to avoid potential fatal reactions. This criterion can be applied to patients who are scheduled to receive or are undergoing radiation therapy.

  This total dose that should not be exceeded (TDNTBE) is expressed in gray (Gy) and is an important parameter for the radiologist. This predicts the maximum dose that any patient can absorb without causing a potentially fatal reaction. This parameter also avoids radiation therapy for patients with particularly high radiosensitivity.

According to the present invention, TDNTBE is
If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / N _pH2AX (t4)], or
If N _pH2AX (t0)> 3, it can be determined by the formula: TDNTBE = 60 / [N _pH2AX (t4) + N _pH2AX (t0)].

In a variation of the method of the invention, for the cell sample, an average number of nuclear lesions (expressed in%) for 100 cells at time t is observed (this average number is referred to as N _MN (t)) and time t is t4 after irradiation with at least t0 (non-irradiation) and absorbed dose D, and the parameter N _MN (t4) is used to determine the TDNTBE. However, because the statistical uncertainty of experimental micronucleus measurements is higher than the statistical uncertainty of the number of nuclear foci observed by immunofluorescence, the predictive value of lesion measurements is the predictive value of micronuclei. It can be said that it is preferable to the numerical value.

Therefore, TDNTBE is
If N _pH2AX (t0) ≦ 3, the formula: TDNTBE = 60 / [0.4 × N _MN (t4)], or if N _pH2AX (t0)> 3, the formula: TDNTBE = 60 / [2+ ( 0.4 × N _MN (t4))].

  Based on this quantitative analysis, a more qualitative diagnosis can be made. This diagnosis is influenced by the quantitative analysis described above, but will take into account any clinical factors that call the physician's attention.

  The correlation between TDNTBE, the number of pH2AX lesions, and the number of micronuclei obtained 24 hours after irradiation with 2 Gy was verified in relation to the number of pH2AX lesions in the natural state. This validation was done in the 1970s with a group of retrospective data collected for highly radiosensitive patients who were irradiated in one or two radiotherapy sessions and had ATM mutations. Death cases after irradiation (severity grade 5 according to the CTCAE classification) have been described in the literature from the 1970s to the present day, systematically, cases of telangiectasia ataxia or ligase 4 Corresponds to the case of a patient with mutation (A. Joubert et al., “DNA double-strand break repair defects in syndromes associated with acute radiation response: At least two different assays to predict intrinsic radiosensitivity?”, Published in the International Journal of Radiation Biology, 2008, vol. 84 (2), p107-125). Based on these retrospective data and considering the total accumulated dose during these radiotherapy sessions, we could contrast the number of corresponding unrepaired double-strand breaks (DSBs). In practice, the number of unrepaired double-strand breaks was counted in a number of dysfunctional lines and for one case of LIG4 mutation (180BR cell line). These cell lines systematically showed a ratio of unrepaired cuts exceeding the lethal limit after a single dose. The values for cell lines from AT and 180BR patients shown in Table 2 include evidence to determine the threshold beyond which the number of unrepaired cuts is fatal to the patient. The CTCAE severity grade corresponding to these special cases is 5 (= death). Therefore, TDNTBE can be defined as the total accumulated dose that can reach this number of unrepaired DSBs.

  In another variation of the method of the invention, a radiosensitive group is first determined for a cell sample.

  The definition of a radiosensitivity group (GROUP) assists physicians in determining similarity to known genetic syndromes from the scores according to the present invention and the clinical picture of the patient. These groups were defined in publications by Joubert et al. Cited above.

-According to the present invention, it is considered as follows:
N _pH2AX (t4) <2, and N _pATM (t1)> N _pATM (t2), and N _pATM (t1)> 30, and A <10, and B <5, and C <2.
Where C = N _pH2AX (t0) + N _MN (t0);
B = percentage of macronuclei (nuclei greater than 150 μm ² ) at t0;
A = number of pH2AX small lesions per cell at N _MRE11 (t0) + t0;
If so, the radiosensitivity group (GROUP criteria) is considered “Group I” and the cell is radioresistant.

_(N pH2AX (t4)> 8 _{or,, N MN (t4)>} 24) if,
The radiosensitivity group (GROUP criteria) is considered “Group III” and the radiosensitivity of the cells in question is high.

  For all other conditions, the GROUP criteria are considered “Group II” and the cells exhibit moderate radiosensitivity.

  After determining the patient's radiosensitivity group, TDNTBE is determined according to one or the other of the above variations.

  In fact, these formulas can be applied to patients belonging to a type II radiosensitivity group (moderate radiosensitivity), patients who can receive standard treatment (radioresistance) (group I), and any It is particularly suitable for determining the TDNTBE of highly radiosensitive patients who do not receive irradiation even under conditions.

EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, these Examples do not limit this application at all. These examples relate to the analysis of patient cell lines and allow determination of the total dose that should not be exceeded.
Example
1. Test Preparation Skin cell samples were taken from patients by biopsy using the “skin punch biopsy” method known to those skilled in the art. The cell sample was placed in DMEM sterile medium supplemented with 20% fetal calf serum. Thereafter, the sample was immediately moved to a specialized laboratory to avoid leaving the cell sample at room temperature for over 38 hours.

  Immediately after moving to the laboratory, cell samples obtained by biopsy were established as a cell line that can be amplified by methods known to culture research institutions and those skilled in the art. In particular, by trypsin dispersion, the cells are diluted again in fresh medium and this is repeated until the desired number of cells is obtained. After obtaining a sufficient number of cells (generally after 1 to 3 weeks), an initial test was performed using the method of the present invention. Cells were seeded on glass slides in petri dishes. A part of the slide glass was irradiated with an absorbed dose D of 2 Gy in a medical irradiator according to an approved dose measurement method. Irradiation was not performed on the other slide glasses, and the natural state was expressed (the absorbed dose was 0 Gy).

Thereafter, the cells on the glass slide were fixed, lysed and hybridized. The cells were fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature, and 20 mM pH 7.4 HEPES buffer (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), NaCl in 50 mM, 3 mM of _MgCl 2, 300 mM sucrose, 0.5% Triton X-100 (formula _{t-Oct-C 6 H 4} - Table with _(OCH 2 CH 2) xOH (wherein x = 9 to 10) And a nonionic surfactant, CAS No. 9002-93-1, manufactured by Sigma Aldrich) for 3 minutes. The cover slide was then washed in phosphate buffered saline (known as PBS) prior to immunological staining. Incubation was performed in PBS supplemented with 2% bovine serum albumin (known as BSA or fraction V; Sigma Aldrich) for 40 minutes at 37 ° C., followed by washing with PBS. Anti-pH2AX primary antibody was used at a concentration of 1: 800, and other primary antibodies were used at a concentration of 1: 100. Incubation with anti-mouse secondary antibody FITC or anti-rabbit secondary antibody TRITC (1: 100, Sigma Aldrich) was performed in 2% BSA at 37 ° C. for 20 minutes.

The slides were then treated with Vectashield ^™ containing DAPI (4,6-diamidino-2-phenylindole) to label nuclei. Even with staining using DAPI, indirectly, the number of cells in resting phase G0 / G1 (cells with uniform DAPI staining), the number of cells in synthetic phase S (cells having many pH2AX lesions), resting It is possible to determine the number of cells in the stage G2 (cells with non-uniform DAPI staining) and the mitotic phase M (state in which chromosomes are visible). Counterstaining with DAPI allowed the localization of nuclei, particularly for localization of cytoplasmic localization or nuclear localization, and thus the quantification of existing micronuclei.

Results were obtained from glass slides using an immunofluorescence microscope (eg Olympus model). Readings were made directly by counting lesions obtained with at least 50 cells in G0 / G1 at each point using different markers pH2AX, pATM, and MRE11, as well as dedicated image analysis software (ImageJ).
2. PH2AX, MRE11, and pATM lesions at 10 minutes (t1), 1 hour (t2), 4 hours (t3), and 24 hours (t4) after post-irradiation repair in the natural state and absorbed dose 2 Gy Number, natural state, and number of micronuclei N _MN (t) (expressed in%) observed for 100 cells at a repair time of 24 hours after irradiation with an absorbed dose of 2 Gy, small pH 2AX per cell in the natural state Determination of the number of lesions and the percentage of macronuclei ( nuclei greater than 150 μm ² ) at t0 For non-irradiated cells (natural state, ie, t0), the average number of natural pH2AX by immunofluorescence analysis of these cells , the number of pH2AX small foci of cells per one natural state, the percentage of large nuclei (larger nuclei than 150 [mu] m ²⁾ at t0, And give the number of micronuclei was observed naturally.

For irradiated cells, radiation irradiation was performed using a medical accelerator that delivers 6 MV photons at an absorbed dose rate of 3 Gy min ⁻¹ . After irradiation with an absorbed dose of 2 Gy, the cells were maintained at 37 ° C. in a culture incubator. For irradiated cells (radiation-induced state), samples are collected after a specified period of time, specifically 10 minutes (t 1), 1 hour (t 2), 4 hours (t 3), 24 hours after cessation of radiation. Labeled after time (t4). Using the markers pH2AX, MRE11, and pATM at each post-irradiation repair time (10 minutes (t1), 1 hour (t2), 4 hours (t3), and 24 hours (t4)) The average number of acquired nuclear lesions was obtained. The number of micronuclei N _MN (t) (expressed in%) per 100 cells observed 24 hours after irradiation with an absorbed dose of 2 Gy was also determined by immunofluorescence analysis of the sample.

  In order to obtain results with sufficient statistical confidence to use as a basis for diagnosis, a series of three irradiations were performed individually. The mean and standard error of the number of lesions in the natural state (t0), 10 minutes after post-irradiation repair (t1), 1 hour (t2), 4 hours (t3), and 24 hours (t4)) SEM). The calculation results for various patient skin cell samples are shown in the following table (see Table 2).

Table 2: Radiosensitivity and TDNTBE determination of a patient. Judgments were made for various skin cell samples of patients in the natural state (t0) and / or 10 minutes (t1), 1 hour (t2), 4 hours (t3), and 24 hours after irradiation with 2 Gy. Number of pH2AX lesions, pATM lesions, and MRE11 lesions after (t4), number of micronuclei observed for 100 cells 24 hours after the repair period after irradiation with natural state and absorbed dose 2 Gy (t4) N _MN (T) (expressed in%), in relation to the natural number of pH2AX small lesions per cell and the percentage of macronuclei (nuclei greater than 150 μm ² ) at t0. ( ^* This patient is radioresistant and can therefore receive a standard radiotherapy protocol: 70 Gy for prostate cancer treatment and 40 Gy for breast cancer treatment. )

2.3 Predictive assessment of the total dose that should not be exceeded To avoid potential fatal reactions in patients scheduled to receive radiation therapy or undergoing radiation therapy for various patient skin cell samples (see Table 2) The total dose that should not be exceeded (TDNTBE) was determined.

The total dose not to be exceeded (TDNTBE) expressed in gray (Gy) is
If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / N _pH2AX (t4)
Or, if N _pH2AX (t0)> 3, it was determined by the formula: TDNTBE = 60 / [N _pH2AX (t4) + N _pH2AX (t0)].

According to another variant of the invention, the total dose not to be exceeded (TDNTBE), expressed in gray (Gy), is expressed as the number of micronuclei per 100 cells observed at time t (expressed in%). )
If N _pH2AX (t0) ≦ 3, TDNTBE = 60 / [0.4 × N _MN (t4)]
Or
If N _pH2AX (t0)> 3, it was also determined by TDNTBE = 60 / [2+ (0.4 × N _MN (t4))].

  Times t0 and t4 correspond to the natural state, i.e., non-irradiation, and time t4 after irradiation with absorbed dose D, respectively.

  Table 2 shows the quantitative values of total dose (TDNTBE) that should not be exceeded, expressed in gray (Gy).

  Severity grades 2-4 correspond to tissue reactions (eg, dermatitis, proctitis, etc.). Severity grade 1 is a tolerable side effect and is often confused with grade 0 (no effect) by some physicians.

Claims (14)

A method for predicting cellular radiosensitivity to ionizing radiation of a cell sample obtained from cells sampled from a non-irradiated or slightly irradiated site of a patient, comprising:
(i) the sampled cells are amplified, and the amplified cells constitute the “cell sample”;
(ii) Using the marker pH2AX, the average number of nuclear lesions obtained at the observation time t is determined for the cell sample, and this average number is _defined as N _pH2AX (t), and the observation time t is 0 minutes That is, t0 representing a non-irradiation state and an observation time t1 after irradiation with an absorbed dose D, preferably at least one selected from t2, t3, and t4,
(Iii) a total dose not to be exceeded (TDNTBE), expressed in gray (Gy), is determined using at least the parameter N _pH2AX (t4);
The t4 is a fixed value representing the time required for the level of DNA cleavage to reach its residual value, and preferably 6 to 8 times t3, but 12 hours or more, preferably 12 Time to 48 hours, more preferably about 24 hours,
The t3 is a fixed value representing the time at which about 25% of the DSB of the control cells collected from the patient having radiation resistance is repaired at the end thereof, and preferably 3 to 5 times t2. However, it is selected to be 2.5 hours or more and 6 hours or less, preferably 3 hours to 5 hours, more preferably about 4 hours,
The t2 is a fixed value representing a time at which about 50% of the DSB of the control cells collected from a patient having radiation resistance is repaired at the end thereof, and preferably 5 to 7 times t1. However, it is selected to be 35 minutes or more and 90 minutes or less, preferably 45 minutes to 75 minutes, more preferably about 60 minutes,
t1 is a fixed value that represents the time at which the number of recognized DSBs reaches a maximum value in the control cells taken from a radiation resistant patient at the end, preferably 5 A method characterized in that it is selected to be between min and 15 min, preferably between 7.5 min and 12.5 min, more preferably around 10 min. In claim 1,
The average number (expressed in%) of the nuclear lesions observed for 100 cells at time t is determined, and this average number is defined as N _MN (t), and the time t is at least non-irradiated t0 and absorbed dose T4 after irradiation at D, and the parameter N _MN (t4) is used to determine TDNTBE. In claim 1 or 2,
TDNTBE
If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / N _pH2AX (t4) or
If N _pH2AX (t0)> 3, it is determined by the formula: TDNTBE = 60 / [N _pH2AX (t4) + N _pH2AX (t0)]. In claim 2 or 3,
The TDNTBE is
If N _pH2AX (t0) ≦ 3, according to the formula: TDNTBE = 60 / [0.4 × N _MN (t4)] or
If N _pH2AX (t0)> 3, it is determined by the formula: TDNTBE = 60 / [2+ (0.4 × N _MN (t4))]. In any one of Claims 1-4,
The method wherein the sampled cells are fibroblasts collected from a patient's skin biopsy. In any one of Claims 1 to 5,
The absorbed dose D is 0.5 Gy to 4 Gy, preferably 1 Gy to 3 Gy, more preferably 1.7 Gy to 2.3 Gy, and still more preferably 2 Gy. In any one of Claims 1-6,
t1 is 8 minutes to 12 minutes, t2 is 50 minutes to 70 minutes, t3 is 3.5 hours to 4.5 hours, and t4 is 22 hours to 26 hours. In claim 6 or 7,
A method wherein t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours, and D is 2 Gy. In any one of Claims 1-8,
The determination of N _pH2AX (t) involves an immunofluorescence test. In any one of Claims 1-9,
A method characterized in that said control cells taken from a patient with radiation resistance are selected as cells whose in vitro clonogenic survival rate is higher than 55% after irradiation with an absorbed dose of 2 Gy. In any one of claims 1 to 10,
The method wherein the control cells collected from a patient having radiation resistance are selected as cells collected from a patient who did not show a significant tissue response during or after radiation treatment. In any one of claims 3 to 11,
The method wherein the average number of nuclear lesions obtained at observation times t1, t2 and t3 using the marker pH2AX is used to verify the shape of the dynamic curve of DSB site recognition. In any one of claims 1 to 12,
A nuclear lesion obtained at observation time t using at least one of the markers pATM and MRE11 and at least one observation time selected from t1, t2, t3 and t4 after irradiation with absorbed dose D A method, characterized in that average numbers are determined and these average numbers are N _pATM (t) and N _MRE11 (t), respectively. In any one of claims 1 to 13,
Prior to TDNTBE, the patient's radiosensitivity group
(A) N _pH2AX (t4) <2, N _pATM (t1)> N _pATM (t2), N _pATM (t1)> 30, A <10, B <5, and C < 2, provided that C = N _pH2AX (t0) + N _MN (t0);
B = percentage of macronuclei (nuclei greater than 150 μm ² ) at t0;
When A = N _MRE11 (t0) + pH 2AX small foci per cell at t0, the sample is considered to be radioresistant (Group I);
_{(B) (N pH2AX (t4} )> 8 _{or,, N MN (t4)>} 24)
The sample is considered to have high radiation sensitivity (Group III),
(C) A method characterized in that, for all other conditions, the sample is determined according to a procedure, which is considered to have moderate radiosensitivity (Group II).